Methods for making intermediates and oxytocin analogues

ABSTRACT

More efficient and/or economical methods for synthesizing heptapeptide alcohol analogs of oxytocin are provided along with novel intermediates which are useful in synthesizing such oxytocin analogs. Efficient and economical methods for synthesizing intermediates useful in synthesizing these oxytocin analogs are also provided.

This application is a divisional of U.S. Ser. No. 10/888,704, filed Jul.8, 2004, which is a continuation of PCT/US03/04301, filed Feb. 13, 2003,which claims priority from U.S. Provisional Application Ser. No.60/360,345, filed Feb. 27, 2002, the disclosures of all of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods for making intermediates andusing same to make equivalent heptapeptide alcohol analogues ofdesamino-oxytocin (i.e. cyclic peptides wherein the N-terminal residueis deaminated and the C-terminus is an alcohol). Improved methods aredisclosed for synthesizing intermediates and for employing theseintermediates to produce the analogues that exhibit oxytocin antagonistactivity and that are useful, inter alia, for decreasing or blockinguterus muscle contraction.

BACKGROUND OF THE INVENTION

Oxytocin is a peptide hormone which stimulates contraction of theuterine muscles, and it is believed to be involved in the etiology ofpre-term labor and dysmenorrhea. Oxytocin antagonists have proved to beuseful in the control of these conditions, and oxytocin antagonistpeptides of good potency and selectivity for therapeutic use aredisclosed in WO 95/02609, published 26 Jan. 1995. They are oftenintended for administration in aqueous solution, and the manufacture ofready-for-use doses of such antagonists may require that such solutionsbe stable for extended periods; which they may not always be. Thepotential need to prepare such a medicament immediately prior to use wasconsidered to be inconvenient and generated an improvement.

U.S. Pat. No. 6,143,722 (EP 938,496; WO 98/23636) discloses equivalentheptapeptide analogues that exhibit oxytocin antagonist activity, whichresemble those disclosed in the earlier WO 95 application, but whereinthe C-terminus of the peptide is reduced to an alcohol. By heptapeptideor equivalent heptapeptide, for purposes of this application, is meant acyclic compound where the N-terminal residue is deaminated and its sidechain is linked by a covalent bond to a side chain of a residue spacedapart therefrom in the peptide chain which contains 6 residues inaddition to the N-terminal residue.

Although such oxytocin antagonist peptides can be synthesized by thesynthesis disclosed in the '722 patent, it requires about 7 separatesteps, counting the peptide synthesis as one and not counting thesynthesis of the modified homocysteine (Hcy) residue. More economicalsyntheses are frequently sought for chemical compounds of potentialcommercial interest, and such is the case in this instance.

SUMMARY OF THE INVENTION

The present invention provides new intermediate compounds and newmethods for making certain intermediate compounds that are useful insynthesizing pharmaceuticals, particularly oxytocin antagonist peptidesof the general type taught in the '722 U.S. patent. The invention alsoprovides improved methods for synthesizing the oxytocin antagonistpeptide alcohols disclosed in the '722 patent, which syntheses permitmore efficient and economical production of such C-terminal alcoholpeptides.

In a more specific aspect, the invention provides an intermediatesuitable for forming a peptide having pharmaceutical properties, whichhas the formula:

wherein P¹ is H or an amino-protecting group; P² is an amino-protectinggroup that is different than P¹ and is not labile under conditions thatwould remove P¹; P³ is H or an amino-protecting group that is differentthan P¹ and is not labile under conditions that would remove P¹,provided however that P² and P³ may be a divalent amino-protectinggroup; n is 2, 3 or 4; R is lower alkyl; and W is H, a protecting groupor resin; and also provides a method for making such an intermediatewherein an α-amino acid having an amino side chain, with its side chainamino group protected and its α-amino group acylated, is reacted in asuitable solvent with a reducing agent to change the acyl group to analkyl group and simultaneously change the α-carboxy group to CH₂OH.

In another specific aspect, the invention provides a method forpreparing an equivalent heptapeptide analogue, or a pharmaceuticallyacceptable salt thereof, having oxytocin antagonist activity andconsisting of a hexapeptide moiety A and a C-terminal β-aminoalcoholresidue B bound to the moiety A by an amide bond, wherein (1) theβ-aminoalcohol B is:

with Q being (CH₂)_(n)-NH₂, with n being 2, 3 or 4, and R being CH₃ orC₂H₅; and (2) the moiety A is:

with X being a D-aromatic α-amino acid, which may optionally have itsside chain protected; and Y being an aliphatic α-amino acid, whichmethod includes the following steps:

-   (a) providing a resin-linked diamino alcohol having the formula:    wherein P¹ is H or an amino-protecting group, Q′ is (CH₂)_(n)-NP²P³,    with n being 2, 3 or 4, P² being an amino-protecting group that is    different from P¹ and not labile under conditions that would remove    P¹, and P³ being H or an amino-protecting group that is the same or    different than P², provided however that P² and P³ may be a divalent    amino-protecting group, and with the resin being one capable of    forming an ether bond with an aliphatic alcohol; (b) N-alkylating to    produce the compound:    wherein R is CH₃ or C₂H₅; (c) adding residues either singularly or    in a group or groups to create the following peptide-resin:    wherein P⁴, P⁵ and P⁶ are individually H or protecting groups; (d)    cleaving from the resin and selectively deprotecting to remove any    protecting groups P⁴, P⁵ and P⁶ to form the linear compound:    (e) cyclizing the linear compound to create the compound:    and-   (f) deprotecting to create the cyclic equivalent hexapeptide:    wherein Q is (CH₂)_(n)-NH₂

In a further more specific aspect, the invention provides a method forpreparing an equivalent heptapeptide analogue, or a pharmaceuticallyacceptable salt thereof, having oxytocin antagonist activity andconsisting of a hexapeptide moiety A and a C-terminal β-aminoalcoholresidue B bound to the moiety A by an amide bond, wherein (1) theβ-aminoalcohol B is:

with Q being (CH₂)_(n)-NH₂, with n being 2, 3 or 4, and R being CH₃ orC₂H₅; and (2) the moiety A is:

and with X being a D-aromatic α-amino acid, which may optionally haveits side chain protected; and Y being an aliphatic α-amino acid, whichmethod includes the following steps: (a) providing a resin-linked aminoacid having the formula:

wherein P⁵ is a protecting group and P⁷ is H or a protecting group;

-   (b) adding residues either singularly or in a group or groups to    create the following peptide-resin:    wherein P⁴ and P⁶ are individually protecting groups;-   (c) cleaving from the resin to form the linear compound:    to form the linear compound:    wherein Q′ is (CH₂)_(n)-NP²P³, with n being 2, 3 or 4, P² being an    amino-protecting group, and P³ being H or an amino-protecting group    that is the same or different than P², provided however that P² and    P³ may be a divalent amino-protecting group;-   (e) selectively deprotecting and cyclizing the linear compound to    create the compound:    and-   (f) deprotecting to create the cyclic equivalent heptapeptide:    wherein Q is (CH₂)_(n)-NH₂.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides improved methods for synthesizing suchequivalent heptapeptide analogues exhibiting therapeutically usefuloxytocin antagonist activity and improved stability in aqueous media.These equivalent heptapeptide analogues are characterized by a structurewhich comprises an N-terminal hexapeptide analogue moiety A and aC-terminal diaminoalcohol moiety B. The structure of the diaminoalcoholmoiety B is:

wherein Q is —(CH₂)_(n)NH₂, with n being 2, 3 or 4,

and R is CH₃ or CH₂H₅.The structure of the moiety A is:

wherein Mpa, Ile, Asn and Abu have the following meanings:

Mpa 3-mercaptopropionic acid residue (otherwise calleddesamino-cysteine)

Ile isoleucine residue

Asn asparagine residue

Abu α-aminobutyric acid residue;

and wherein

X is a D-aromatic α-amino acid residue; and

Y is an aliphatic α-amino acid residue.

By an aromatic α-amino acid is meant an α-amino acid wherein the sidechain includes an aromatic ring system. Such a system may be carbocyclicor heterocyclic, monocyclic or fused. Examples of aromatic α-amino acidsinclude (but are not limited to) phenylalanine, tyrosine,(O-ethyl)tyrosine, tryptophan, β-(2-naphthyl)alanine and phenylglycine,and the residue X is of the unnatural D-configuration.

By an aliphatic α-amino acid is meant an α-amino acid of the naturalL-configuration wherein the side chain has only carbon and hydrogenatoms. Such side chains may include alkyl and cycloalkyl groups; theymay be unsaturated but may not include aromatic residues. The sidechains may have 1 to 12 carbon atoms, although the preferred range is3-7 carbon atoms. Examples of aliphatic α-amino acids include alanine,valine, leucine, isoleucine, alloisoleucine (aIle), cyclohexylglycine,(β,β-diethyl) alanine and adamantylalanine.

In the structure of the hexapeptide analogue moiety A, the line joiningthe Mpa and Abu residues has its conventional meaning, signifying thatthere is a covalent bond linking the ends of the side chains of thesetwo residues. In this case, a sulfur atom at the end of the side chainof the N-terminal Mpa residue is joined by a covalent bond to the γ- (or4-) carbon atom of the Abu residue side chain.

The diaminoalcohol moiety B includes a stereogenic centre, so it canexist in two epimeric forms, R and S, corresponding to the D and Lisomers of the related amino acids. Heptapeptide analogues with eitherof these isomers are acceptable, as are mixtures of epimers. Preferably,the diaminoalcohol moiety is present as a single epimer, and morepreferably it has the S configuration.

In the context of the present application, the Mpa residue and thediaminoalcohol B are considered to be equivalents of α-amino acids;thus, the compounds of interest are termed heptapeptides or equivalentheptapeptides.

In preferred compounds, X is either a D-tryptophan residue or aβ(2-naphthyl)-D-alanine residue, Y is a residue of valine, leucine,isoleucine, alloisoleucine, cyclohexylalanine, or (β,β-diethyl)alanine.

Some of the particularly preferred compounds are the following:

wherein the following further abbreviations have been used:

D-Trp D-tryptophan residue

aIle alloisoleucine residue

Ala(3,3-diethyl) (β,β-diethyl)alanine residue

D-Nal β-(2-naphthyl)-D-alanine residue

Leu leucine residue

Val valine residue

The first peptide listed above is presently the most preferred compound.

These peptides of interest contain a basic site (amine) and so can formsalts with acids, which salts retain the pharmacological properties ofthe free bases. Examples of such salts include (but are not limited to)the hydrochloride, hydrobromide, sulfate, acetate, citrate, benzoate,trifluoroacetate and methanesulfonate. The methods disclosed are usefulsteps in producing pharmaceutical compositions which include apharmacologically effective amount of at least one of the oxytocinantagonist heptapeptide analogues described above. Such compositions mayinclude pharmaceutically acceptable additives or carriers, such aspreservatives, diluents, dispersing agents, agents to promote mucosalabsorption, buffering agents and flavorings, such as disclosed in the'722 patent and may be so administered for reducing or blocking thecontraction of the uterine muscle. One preferred composition is asterile aqueous solution of such a heptapeptide analogue in isotonicsaline, which is suited to intranasal administration or intravenousinjection, containing a buffering agent, e.g. a phosphate/citratebuffer, to maintain the pH of the solution in the range 0.3-7.0, andpreferably in the range 3.5-5.5. With respect to routes ofadministration, intravenous or subcutaneous injections are likely to bethe most efficient routes of delivery, while intranasal administrationcan be expected to be more efficient than oral dosing. Generally, theamount of compound constituting a single effective dose for intravenousor subcutaneous treatment of an average woman in pre-term labor is fromabout 0.1 mg to about 500 mg, and preferably from about 1 mg to about200 mg, in a period of 24 hours. The invention provides improved methodsof synthesizing such oxytocin antagonist heptapeptide analogues andintermediates for use in such syntheses, which methods are moreeconomical than prior art methods and/or have higher yields.

These overall methods for preparing a heptapeptide analogue of interest,or a pharmaceutically acceptable salt thereof, having oxytocinantagonist activity generally comprise making a compound that consistsof a hexapeptide moiety A and a C-terminal diaminoalcohol residue B thatis initially or eventually bound to the moiety A by an amide bond,

wherein (1) the diaminoalcohol B is:

with Q being (CH₂)_(n)-NH₂, with n being 2, 3 or 4, and R being CH₃ orC₂H₅; and

(2) the moiety A is:

Some key intermediates that may be employed in the overall syntheses ofinterest include the α-alkylated diamino alcohol either as the free orprotected alcohol or attached to a suitable resin by an ether bond, andthe known compound “carba-6” described hereinafter. The invention alsoprovides methods that are useful to produce these key intermediateswhich then can be effectively employed in efficient syntheses of theseheptapeptide analogues.

The improved overall syntheses that have been developed for making thesecyclic heptapeptide oxytocin antagonists result in simplification ofsteps, increased yields and/or lower costs of raw materials, thusrendering them much more desirable routes to the compounds of choice.These syntheses generally utilize such an alkylated diamino alcohol andthe protected amino acid referred to as Fmoc-carba-6, i.e.,

The intermediate Fmoc-carba-6 may be synthesized using astate-of-the-art method as disclosed in the '722 patent; however, animproved synthesis for this intermediate is hereinafter described.

The known synthesis for the intermediate Fmoc-carba-6, i.e.Fmoc-Abu(SCH₂CH₂CO₂t-Bu)OH that is disclosed in the '722 patent startswith the dimer of homocysteine, (Hcy)₂. However, the cost of thisstarting material is considerable, i.e. currently over about $250 for 5grams. An improved synthesis for this intermediate, i.e., Fmoc-carba-6,has been developed which allows it to be economically synthesizedthrough the use of readily available methionine (Met) as a startingmaterial. The cost difference of starting materials is highlysignificant because the cost per mol of Met is about 50 times less.Methionine is first reduced by treatment with sodium in ammonia, and theresultant sulfhydryl group is then alkylated using either t-butylacrylate or t-butyl 3-bromopropionate to add the moiety that eventuallybecomes the N-terminal residue of the heptapeptide. At this point, anN^(α)-protecting group is routinely introduced. In addition to saving onraw material cost, it was found that, when performed on a large scale,the process results in a product that can be precipitated in crystallineform from a solution of ethyl acetate during evaporation, thussimplifying the purification process.

A novel intermediate used to build the C-terminal portion of theultimate linear hexapeptide is an N-protected or unprotected, N-alkyl,β-amino alcohol derived from a protected α-amino acid where theα-carboxyl group has been transformed to an alcohol and may optionallybe protected by a protecting group or be coupled to a resin. In otherwords, this intermediate is a diaminoalcohol which is defined to by theformula:

wherein P¹ is H or an amino-protecting group; P² is an amino-protectinggroup that is different than P¹ if P¹ is present and is not labile underconditions that would remove P¹; P³ is H or an amino-protecting groupthat is different than P¹ and is not labile under conditions that wouldremove P¹, provided however that P² and P³ may be a divalentamino-protecting group; n is 2, 3 or 4, R is alkyl; and W is H, aprotecting group or resin. A suitable resin, such as a chlorotritylresin, may be used, and the optional protecting agent may be one ofthose commonly used to protect Ser or Thr, e.g. TMS (trimethylsilyl). Ris preferably methyl or ethyl and more preferably is methyl. Theintermediates used in making the preferred cyclic equivalentheptapeptides are those wherein n is 3, i.e. the protected N^(α)-methylornithinol; thus, this compound is a particularly preferredintermediate.

Briefly, it has been found that such a methyl ornithine alcohol can beefficiently synthesized by beginning with Orn, the side chain aminogroup of which is protected by benzyloxycarbonyl (Z) or by anothersuitable amino-protecting group that remains when the α-amino protectinggroup is removed; H-Orn(Z)-OH is a commercially available startingmaterial. Other protected amino acids having either 2 or 4 methylenegroups in the amino side chain can alternatively be employed to producea different desired end product. Treatment with formic acid and aceticanhydride converts the α-amino group to a formamido moiety. Thereafter,treatment with boron hydride complexed in tetrahydrofuran reduces theα-carboxyl group to CH₂OH and the formamide moiety to methylamino. If anethylamino group is desired, the α-amino group of Orn is insteadacetylated to add an acetyl group rather than a formyl group.Thereafter, if desired the α-amino group can be protected e.g. withFmoc, and the compound can be coupled to a resin by an ether bond at thealcohol. Alternatively, Fmoc-Orn(Z)-ol may be linked to a 2Cl-Trt resin,as by reaction in DCM containing pyridine, and used to build ahexapeptide on the resin; Fmoc-Orn(Z)-ol is easily obtained fromcommercially available Fmoc-Orn(Z)-OH by reduction of its mixedanhydride with sodium borohydride.

In one overall synthesis for making the desired cyclic equivalentheptapeptides, Fmoc-carba-6 is linked to a 2-chlorotrityl chloride resin(2Cl-Trt resin) by reaction with the resin to form an ester bond withthe α-carboxyl group while the side chain omega carboxy group and theα-amino group are protected. Other suitable resins might alternativelybe used. The Fmoc protection is then removed from the α-amino group, anda pentapeptide is created by sequentially coupling with Fmoc-Asn,Fmoc-Y, Fmoc-Ile and then Boc-X. The side chains of Asn, X and/or Y maybe protected if desired. For example, when X is D-Trp, it may bedesirable to protect the indole nitrogen; however, the synthesis can beeffectively performed without protecting either Asn or D-Trp. The use ofBoc-protection for the last amino acid to be added allows for the latersimultaneous cleavage of the t-butyl ester group protecting the sidechain carboxy and the N-terminal protecting group.

At this point in the synthesis, the linear peptide is cleaved from theresin (as by treatment with a mixture of DCM/TFE/AcOH) to produce thepentapeptide having a free acid at its C-terminus. A reaction is thencarried out to add H-MeOrn(Z)-ol at the C-terminus, preferably by amixed anhydride method to reduce potential racemization of the Aburesidue. If desired, the alcohol can be protected with a suitableprotecting group, e.g. trityl, TMS or benzyl ether; for example, thereaction was successfully carried out after N^(α)MeOrn(Z)-ol wasconverted to its N,O-bis (trimethylsilyl) derivative by the treatmentwith N, O-bis (trimethylsilyl) acetamide. However, it is felt that sucha step is clearly optional and is not needed to protect againstO-acylation. After this addition to create the hexapeptide is effected,deprotection is carried out using TFA in dichloromethane (DCM) withsuitable scavengers, to simultaneously remove the Boc protection at theN-terminus and the t-butyl protection of the side chain carboxyl group.Cyclization is then effected to link the long side chain to the α-aminogroup of D-Trp and thereby create the equivalent heptapeptide,preferably being suitably carried out, e.g. in the presence of PyBOP andDIPEA in dimethyl formamide (DMF). Thereafter, the final deprotection ofthe side chain amino group of ornithinol is carried out using hydrogenfluoride or trimethylsilyl bromide with suitable scavengers to producethe cyclic, C-terminal alcohol, oxytocin antagonist equivalentheptapeptide, which can be purified by HPLC and converted to the acetatesalt by ion exchange.

In an alternative overall synthesis, the desired cyclic equivalentheptapeptide alcohol is efficiently made by initially linkingcommercially available Fmoc-Orn(Z)-ol to a 2Cl-Trt resin as by reactionin pyridine. Thereafter, the Fmoc protection is suitably removed bytreatment with an appropriate base, such as piperidine, and the α-aminogroup is alkylated on the resin, as by treatment first with o-NBS-Cl and2,4,6-collidine in DCM, and then with the addition of a mixture of TPP,DIAD and MeOH. Various alkyl groups can be introduced by employingdifferent alcohols. N-methylation can also be achieved by treating theo-NBS resin-bound alcohol with 3-5 eq. of methyl 4-nitrobenzenesulfonate and an appropriate base, such as MTBD. After alkylation iscomplete, final treatment with 2-mercaptoethanol and DBU in DMF removesthe o-NBS. The resulting N^(α)MeOrn(Z)-resin is then sequentiallytreated to stepwise construct the hexapeptide on the resin. The peptidecan be built generally as indicated above, with the first coupling beingwith Fmoc-carba-6. By using DIC/HOBt coupling, protection of the sidechain of Asn can usually be omitted. The Boc-hexapeptide-resin may thenbe cleaved and simultaneously selectively deprotected to remove the Bocand t-Bu protecting groups, as by treating with an aqueous solution ofTFA and DCM containing TIS. Final cyclization to create the cyclicequivalent heptapeptide and deprotection may then carried out as knownin the art. The product is isolated and purified using standardtechniques, and only a single purification step, followed byion-exchange, is required. It can be seen that, by taking advantage ofthe two intermediates, the overall synthesis is greatly simplified andcan be economically performed in high yield.

More specifically, the just-described synthesis would generally includethe following steps:

-   (a) providing a resin-linked diamino alcohol having the formula:

wherein P¹ is H or an amino-protecting group, such as Fmoc or o-NBS, Q′is (CH₂)_(n)-NP²P³, with n being 2, 3 or 4, P² being an amino-protectinggroup that is different from P¹ and not labile under conditions thatwould remove such a P¹ protecting group, and P³ being H or anamino-protecting group that is the same or different than P², providedhowever that P² and P³ may be a divalent amino-protecting group, andwherein the resin is one capable of forming an ether bond with analiphatic alcohol;

-   (b) replacing P¹ with o-NBS if P¹ is present as other than o-NBS or    adding o-NBS, and then N-alkylating to produce the compound

wherein R is CH₃ or C₂H₅;

-   (c) removing o-NBS and adding residues either singularly or in a    group or groups to create the following peptide-resin:    wherein-   P⁴, P⁵ and P⁶ are individually H or protecting groups and X and Y    are as defined above;    Note: This compound may also be written as:-   (d) cleaving from the resin and simultaneously selectively    deprotecting to form the linear compound:-   (e) cyclizing the linear compound to create the compound:-   (f) deprotecting to create the cyclic equivalent heptapeptide:

The following specific examples describe detailed syntheses of compoundsof interest according to the foregoing general outline. They contain thebest modes of these syntheses and are considered to be representative ofthe synthesis of compounds of interest in accordance with the presentinvention; however, they should not be construed to constitutelimitations upon the invention which is defined in the claims appendedhereto.

The following abbreviations are used:

-   TBTU 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium    tetrafluoroborate-   PyBOP benzotriazole-1-yl-oxy-tris-pyrrolidino phosphonium    hexaflurophosphate DIPEAN,N-diisopropyl ethyl amine-   DIC 1,3-diisopropyl carbodiimide-   Boc tert-butyloxycarbonyl-   Z benzyloxycarbonyl-   Fmoc 9-fluorenylmethyloxycarbonyl-   o-NBS-Cl o-nitrobenzenesulfonyl chloride-   TFA trifluoroacetic acid-   DCM dichloromethane-   EDT ethanedithiol-   DMF dimethylformamide-   THF tetrahydrofuran-   MTBD 7-methyl-1,5,7-triazabicyclo[4,4,0]dec-5-ene-   BH₃ THF borane-tetrahydrofuran complex-   DME 1,2 dimethoxyethane-   TIS triisopropylsilane-   DBU 1,8-diazabicyclo[5.4.0]undec-7-ene-   MeCN acetonitrile-   TPP triphenylphosphine-   TFE trifluoroethanol-   TEAP triethylammonium phosphate-   EtOAc ethyl acetate-   DIAD diisopropyl azodicarboxylate-   TMSBr trimethylsilyl bromide-   AcOH acetic acid-   MeOH methanol-   NMM N-methymorpholine-   Ac₂O acetic anhydride-   Et₂O ethyl ether-   Ac₂O acetic anhydride-   FmocONSu N-(9-fluorenylmethoxycarbonyloxy)succinimide

EXAMPLE I Synthesis of N^(α)MeOrn(Z)-ol

444.7 g (1.67 mole) of commercially available H-Orn(Z)-OH was dissolvedin 3.5 L (93 moles) of formic acid. The solution was cooled in an icebath, and 1.17 L (12 moles) of acetic anhydride was subsequently addedin dropwise fashion over two hours while the temperature of the reactionmixture was maintained between about 5° and 15° C. The resultantreaction mixture was stirred at room temperature for about two hoursafter the addition was completed, and the reaction vessel wasrefrigerated overnight. 1.5 L of cold water was then added, and thesolution was concentrated to dryness under reduced pressure. Theresidual, light yellow syrup was taken up into EtOAc, washed with waterand then with saturated NaCl, and then dried overnight over sodiumsulfate. After filtration and evaporation to dryness, the residual syrupwas dissolved in 5 L of ethyl ether. The solution was cooled in a dryice bath and seeded. The resulting crystalline product was filtered,washed with ethyl ether, air dried for two hours and finally dried in alyophilizer. 431.6 g (1.47 mole, 88%) of For-Orn(Z)-OH was obtained.

353.2 g (1.2 mole) of dry For-Orn(Z)-OH was placed in a 10 Lthree-necked round-bottom flask equipped with a condenser and anexternal ice bath. 4 L (4 mole) of a reducing agent, 1 molar BH₃THF inTHF, was added over 30 minutes. An exothermic reaction took placeimmediately with the release of hydrogen gas. A clear, refluxingsolution resulted, and the reaction mixture was stirred overnight.Because the reaction was incomplete (HPLC) analysis), an additional 1 L(1 mole) of 1 molar BH₃THF in THF was added, and the mixture was stirredat room temperature for about 2 hours, at the end of which period, thecompletion of the reaction was evident. The reaction was then quenchedwith 1 L of methanol, and the solvents were evaporated. The residue wasredissolved in 600 mL of methanol and evaporated. The residual oil waspartitioned between 1.5 L of water and 0.8 L of DCM, and the pH wasadjusted to about 2 with HCl. The water phase was separated and washedwith DCM (3×0.8 L), and subsequently the pH was adjusted to 12 with 5molar NaOH. The product was extracted with DCM (3×1.5 L). The combinedextracts were washed with aqueous NaCl and dried over sodium sulfate.The drying agent was filtered off, and the solvent was evaporated. Theresidue was taken up in 0.5 L of ethyl ether, and seed crystals wereadded. 149 g (0.56 mole, 47%) of a white crystalline product wasobtained, which product upon analysis was confirmed to be the desiredN^(α)MeOrn(Z)-ol.

The foregoing shows that reduction using BH₃THF complex was effective toreduce both the formyl group to methyl and the α-carboxyl group toCH₂OH.

EXAMPLE II Synthesis of Carba-6

The modified and protected homocysteine residue that initially formsresidue 5 of the hexapeptide is sometimes referred to “carba-6”. Asindicated hereinbefore, it has the chemical formulaFmoc-Abu(SCH₂CH₂CO₂t-Bu)OH. An efficient synthesis has been designedbeginning with commercially available methionine.

Ammonia (5 L) was condensed into a flask containing 2 moles of Met.About 6.15 moles of sodium were added piece by piece to create thesulfhydyl group at the end of the side chain. Addition was continueduntil blue color persisted for 20 minutes. Ammonium chloride (2.1 mol)was added to the reaction mixture to neutralize sodium amide formed. Theresulting clear solution was left overnight under a stream of nitrogento remove ammonia. The solid residue (L-homocysteine and inorganicsalts) was dissolved in 15 L of degassed water, and the solution wasadjusted to pH 8.1 with hydrochloric acid under nitrogen. To theresulting solution t-butyl acrylate (4 mol) was added dropwise, and thereaction mixture was stirred overnight under nitrogen. The solid productformed was collected by filtration, washed with water and t-butyl-methylether to give crude S-(2-tertbutoxycarbonylethyl)-homocysteine, i.e. itssulfhydryl group being substituted with a protected propionyl group. Thecrude product (1.53 mol) was suspended in water, and saturated sodiumbicarbonate solution was then added followed by THF. Then 5 M NaOH wasdelivered from an autotitrator, at pH not exceeding 10, until a clearsolution was obtained. To this solution, a suspension of FmocONSu (1.68mol) in THF was added portionwise while pH, after an initial drop, waskept at 7.5-8 by delivering 5 M NaOH from autotitrator. The reactionmixture was stirred for 4 hours and left in a refrigerator overnight.THF was evaporated, and the residue was extracted with ether. Threelayers were formed. The middle one, which was found to contain thedesired product, was collected. It was determined by HPLC that themiddle layer contained 5-10% of the major by-product (Fmoc-Hcy)₂. Toconvert it to the desired product, the middle layer was diluted withwater, tributylphosphine was added to the resulting solution, and thereaction mixture was stirred for 45 minutes followed by extraction withether. To the aqueous layer t-butyl acrylate was added, and the mixturewas stirred overnight. After washing with ether, the aqueous solutionwas acidified to pH 2 with 6 M HCl, and the product was extracted withethyl acetate. The extract was dried and concentrated in vacuo. Uponevaporation of ethyl acetate, the product, Fmoc-carba-6, started tocrystallize. At this point, the concentrated ethyl acetate solution wasdiluted with ether and then with hexane. The precipitate was collected,washed with a 1:1 mixture of ether and hexane and dried in vacuo to givethe final product as a white solid. More product was recovered from themother liquors to provide a total (from Met) of 625.6 g (1.29 moles) fora 64% yield.

The procedure is straightforward, gives good yields and good purityafter a single crystallization. There is no need for preparing a DCHAsalt intermediate as described in the literature.

EXAMPLE III Synthesis of Cyclic Heptapeptide

One improved synthesis of these heptapeptides of interest initiallybuilds a pentapeptide on a 2Cl-Trt resin and is described hereinafter.

Fmoc-protected carba-6 was coupled to a resin by nucleophilicdisplacement of the chlorine ion in the presence of DIPEA. Cleavage ofthe Fmoc group was then carried out using 25% piperidine in DMF for 30minutes. Thereafter, the next four amino acids, e.g. Asn, aIle, Ile andD-Nal were sequentially added, each time using about a 2.5 molar excessof Fmoc-AA (or Boc-AA for the N-terminus) and converting the amino acidto its HOBt ester and using an equimolar amount of DIC; coupling tookplace at room temperature over about 2 hours. Upon completion of thepentapeptide, cleavage from the resin took place by treatment with amixture of acetic acid, TFE and DCM, at a ratio of 1 part/2 parts/7parts, for about 1½ hours at room temperature, leaving the protectinggroups in place, which facilitate the next coupling at the C-terminus.

Next the pentapeptide was coupled with N^(α)MeOrn(Z)-ol (see Example I)using the mixed anhydride method in order to reduce potentialracemization of the Abu residue. The mixed anhydride was generated fromthe peptide and isobutyl chloroformate in the presence of NMM, andcoupling readily proceeded. The crude hexapeptide was isolated byaqueous workup and lyophilized from t-butanol.

The crude hexapeptide was then treated with TFA/DCM(1:1) containingabout 2% of TIS, for about 1 hour, to remove the Boc-protecting group atthe N-terminus and the t-butyl based protecting group on the Abu residueside chain. After evaporation of the solvents, it was lyophilized froman aqueous mixture containing about 60% MeCN and about 0.1% TFA.Cyclization was then carried out in DMF at the concentration of about1.5 millimole per liter of the crude peptide, using PyBOP in thepresence of DIPEA. After completion of the reaction, the solvents wereevaporated, and the residue was lyophilized from t-butanol. Removal ofthe Z-protecting group was then accomplished using HF at 0° C. for about1 hour. The crude peptide was then precipitated with ethyl ether, andlyophilized from an aqueous solution of acetonitrile.

Final purification of the cyclic equivalent heptapeptide was carried outon a C18 HPLC cartridge using a TEAP system at a pH of about 2.3. Thefractions containing the pure peptide were pooled and reapplied to a C18cartridge, which was then washed with about 5 volumes of 0.1 molarammonium acetate in order to obtain the acetate salt. Final elution wascarried out with 2% acetic acid/acetonitrile system, and the fractionscontaining the pure product were pooled and lyophilized. Massspectrometry (electro spray ionization, ion trap analysis, positivemode) indicated a molecular mass in agreement with the calculated massof the expected structure, i.e. measured m/z equals 841.5; calculatedm/z equals 841.5.

The overall method produces high yield, and the final purification isstraightforward. As an alternative to deprotecting with HF, one mightuse a cocktail of TMSBr/thioanisole/TFA/EDT(1:1:6:0.5).

Oxytocin Receptor Binding Assay

Recombinant human oxytocin receptors were expressed in CHO cells usingstandard molecular biological techniques. A membrane fraction wasprepared and incubated in the presence of [¹²⁵I]-oxytocin and varyingconcentrations of equivalent heptapeptide analogue. Membranes were thenisolated by filtration and counted for radioactivity to determineoxytocin binding. An inhibition constant K_(i) was determined for theanalogue. The results obtained showed a value of 0.1 nM, which isconsidered to be excellent. Similar results were obtained using suchreceptors expressed in HEK293 cells.

EXAMPLE IV Alternative Synthesis of Cyclic Equivalent Heptapeptide

Another synthesis for these equivalent heptapeptide alcohols was derivedin order to further simplify the overall procedure and to avoid the needto prepare MeOrn(Z)-ol or Fmoc-MeOrn(Z)-ol. The synthesis initiallydirectly attaches Fmoc-Orn(Z)-ol to 2-Cl-Trt resin. The hexapeptide issubsequently assembled, and it is cleaved as a linear peptide andsimultaneously selectively deprotected and thus is ready to be cyclized.This approach reduces the risk of racemization as a G+1 coupling is notnecessary.

1) Synthesis of Fmoc-Orn(Z)-ol

Commercially available Fmoc-Orn(Z) was converted to the alcohol byreduction of its mixed anhydride with sodium borohydride. Aftercrystallization from EtOAc, the compound was thereafter used withoutfurther purification.

2) Attachment of Fmoc-Orn(Z)-ol to resin.

1.06 g of 2-Cl-Trt resin (1.39 mmol) was preswollen in DCM, and asolution of Fmoc-Orn(Z)-ol (0.84 mmol) and 5 eq of pyridine in 10 ml ofDCM was added. The disappearance of the alcohol was monitored by HPLC todetermine the progress of the reaction.

A sample of the reaction mixture was saved to assess the stability ofthe Fmoc group under the reaction conditions. The Fmoc group was foundto be sufficiently stable under the conditions used. The reaction wasallowed to proceed for approximately 36 hours to achieve substitution ofapproximately 0.5 mmol/g of 2-Cl-Trt resin.

3) Alkylation on the resin.

The Fmoc group was then removed with 25% piperidine in DMF. The compoundwas first treated with o-NBS-Cl in the presence of 2,4,6-collidine inDCM to convert the α-amino group into a sulfonamido group that issuitably reactive. After 1 hour, the ninhydrin test was negative, andthe resin was then washed with DMF and DME. The resin-bound sulfonamidewas then treated with MeOH/DIAD/TPP, 10 equivalents, overnight, usingDME as a solvent, causing the amino group to be alkylated. Aliquots ofthe resin before and after alkylation were cleaved with 50% TFA/DCM, andthe samples were analyzed by HPLC. Alkylation was deemed to be complete,and the o-NBS group was removed by treatment with 2-mercaptoethanol andDBU in DMF.

4) Building the linear peptide.

Five residues were then sequentially added as in Example III,substituting Boc-D-Trp for Boc-D-Nal. No HOBt was used in the couplingof Fmoc-carba-6.

5) Cyclization of the Equivalent Heptapeptide.

The hexapeptide was cleaved from the resin and simultaneouslyselectively deprotected by treatment with TFA/DCM (1:1) and 1% of TIS toproduce the compound H-D-Trp-Ile-aIle-Asn-Hcy((CH₂)₂COOH)-MeOrn(Z)-ol.Cyclization was then carried out as in Example III by treatment withPyBOP and DIPEA in DMF. Once cyclization was complete, the finalprotecting group was removed by treatment with a TFA/TMSBr/thioanisolecocktail. Purification was then carried out as in Example III usingHPLC, and the peptide was converted to its acetate salt. Massspectrometry indicated a molecular mass in agreement with the calculatedstructure, i.e. measured m/z equals 830.5; calculated ni/z equals 830.5.

This procedure employs a minimum number of steps and a singlepurification and is considered to be highly efficient and economical. Aninhibition constant K₁ for binding in the oxytocin receptor assay wasdetermined and found to be 0.1 nM, essentially the same as for thepeptide resulting from Example III, confirming the apparentinterchangeability of the D-Trp and D-Nal residues in these analogues.

Although the foregoing description and the examples provide the bestmode presently known to the inventors for carrying out their invention,it should be understood that various changes and modifications as wouldbe apparent to one skilled in this art may be made without departingfrom the scope of the invention which is set forth in the claimsappended hereto. For example, although the description has been focusedon certain preferred compounds, particularly ones which incorporate aderivative of ornithine at the C-terminus, it should be understood thatsimilar α-amino acids having longer or shorter side chains might beemployed if desired without departing from the process. The disclosuresof the previously enumerated patents are expressly incorporated hereinby reference.

Particular features of the invention are emphasized in the claims thatfollow.

1. A method of preparing L-homocysteine (Hcy) having an extended andprotected side chain and having its α-amino group protected, whichmethod comprises the steps of: adding Na to a solution of L-methioninein ammonia to form sodium amide and Hcy, removing ammonia and sodiumamide and alkylating the sulfhydyl group of the Hcy side chain with amoiety that terminates in protected carboxy group, and adding aprotecting group to the α-amino group of Hcy that is not labile underconditions that would remove said carboxy-group protection.
 2. Themethod of claim 1 wherein the α-amino group is protected with abase-labile protecting group.
 3. The method of claim 2 wherein theα-amino group is protected with Fmoc.
 4. The method of claim 1 whereinthe sulfhydryl group is alkylated with a moiety which contains anacid-labile protecting group.
 5. The method of claim 4 wherein thesulfhydryl group is alkylated with a moiety which contains a t-butylprotected carboxyl group.
 6. The method of claim 5 wherein thesulfhydryl group is alkylated with —CH₂—CH₂—COO-tBu and the α-aminogroup is protected with Fmoc.
 7. The method of claim 6 wherein theresultant compound is coupled to a resin via the α-carboxy group andthereafter, following removal of said Fmoc protecting group, amino acidsare coupled to form a pentapeptide on said resin.
 8. The method of claim7 wherein the resultant pentapeptide is cleaved from the resin andcoupled at its C-terminus to an N^(α)methyl α-amino alcohol to form ahexapeptide.
 9. The method of claim 8 wherein the resultant hexapeptideis reacted to deprotect said side chain carboxy group and cyclize it byforming an amide bond with the α-amino group of the residue at theN-terminus of said hexapeptide to create an equivalent heptapeptide. 10.The method of claim 1 wherein the resultant compound is coupled to aresin via the α-carboxy group and thereafter, following removal of saidα-amino protecting group, amino acids are coupled to form a pentapeptideon said resin.
 11. The method of claim 10 wherein the resultantpentapeptide is cleaved from the resin and coupled at its C-terminus toan N^(α)methyl α-amino alcohol to form a hexapeptide.
 12. The method ofclaim 11 wherein the resultant hexapeptide is reacted to deprotect saidside chain carboxy group and cyclize it by forming an amide bond withthe α-amino group of the residue at the N-terminus of said hexapeptideto create an equivalent heptapeptide.
 13. A method for preparing aheptapeptide analogue, or a pharmaceutically acceptable salt thereof,having oxytocin antagonist activity and consisting of a hexapeptidemoiety A and a C-terminal β-aminoalcohol residue B bound to the moiety Aby an amide bond, wherein (1) the β-aminoalcohol B is:

with Q being (CH₂)_(n)-NH₂, with n being 2, 3 or 4, and R being CH₃ orC₂H₅; and (2) the moiety A is:

with Mpa, Ile, Asn and Abu having the following meaning: Mpa3-mercaptopropionic acid residue Ile isoleucine residue Asn asparagineresidue Abu α-aminobutyric acid residue; and with X being a D-aromaticα-amino acid, which may optionally have its side chain protected, and Ybeing an aliphatic α-amino acid; which method includes the followingsteps: (a) providing a resin-linked amino acid having the formula:

wherein P⁵ is a protecting group and P⁷ is H or a protecting group; (b)adding residues either singularly or in a group or groups to create thefollowing pentapeptide-resin:

wherein P⁴ is H or a protecting group and P⁶ is a protecting group; (c)cleaving from the resin to form the linear free acid pentapeptide:

(d)

to the product of step (c) to form the linear hexapeptide:

wherein Q′ is (CH₂)_(n)-NP²P³, with n being 2, 3 or 4, P² being anamino-protecting group, and P³ being H or an amino-protecting group thatis the same or different than P², provided however that P² and P³ may bea divalent amino-protecting group; (e) selectively deprotecting andcyclizing the linear compound resulting from step (d) to create theequivalent heptapeptide:

and (f) deprotecting to create the cyclic equivalent heptapeptide:


14. The method of claim 13 wherein X is D-Trp or D-Nal, Y is aIle and nis
 3. 15. A method for preparing an equivalent heptapeptide analogue, ora pharmaceutically acceptable salt thereof, having oxytocin antagonistactivity and consisting of a moiety A and a C-terminal β-aminoalcoholresidue B bound to the moiety A by an amide bond, wherein (1) theβ-aminoalcohol B is:

with Q being (CH₂)_(n)-NH₂, with n being 2, 3 or 4, and R being CH₃ orC₂H₅; and (2) the moiety A is:

with Mpa, Ile, Asn and Abu having the following meaning: Mpa3-mercaptopropionic acid residue Ile isoleucine residue Asn asparagineresidue Abu α-aminobutyric acid residue; and with X being a D-aromaticα-amino acid, which may optionally have its side chain protected, and Ybeing an aliphatic α-amino acid; which method includes the followingsteps: (a) reacting an α-amino acid having a protected amino side chainand an acylated α-amino group with a reducing agent to change the acylgroup to an alkyl group and to simultaneously change the α-carboxy groupto CH₂OH to provide a diamino alcohol having the formula:

wherein P¹ is H or an amino-protecting group, Q′ is (CH₂)_(n)-NP²P³,with n being 2, 3 or 4, P² being an amino-protecting group that isdifferent from P¹ and not labile under conditions that would remove P¹,and P³ being H or an amino-protecting group that is the same ordifferent than P², provided however that P² and P³ may be a divalentamino-protecting group; (b) reacting said product of step (a) with thepentapeptide:

wherein P^(4,) P⁵ and P⁶ are individually protecting groups to form thelinear hexapeptide:

and c) selectively deprotecting and cyclizing the linear compound tocreate the heptapeptide:


16. The method of claim 15 wherein, in step (a), said α-amino acidhaving the protected amino side chain and the acylated α-amino group isreacted with a reducing agent to change the acyl group to an alkyl groupand to simultaneously change the α-carboxy group to CH₂OH.
 17. Themethod of claim 15 wherein, in step (a), ornithine, having its sidechain amino group protected and its α-amino group formylated, is reactedwith a reducing agent in a suitable solvent to simultaneously change theformyl group to a methyl group and to change the α-carboxyl group toCH₂OH.
 18. The method of claim 15 wherein, prior to step (a), ornithine,having its side amino chain group protected, is reacted with formic acidand acetic anhydride to formulate the α-amino group, and thereafter instep (a) the resultant product is reacted with a reducing agent in asuitable solvent to simultaneously change the formyl group to a methylgroup and to change the α-carboxyl group to CH₂OH.
 19. The method ofclaim 18 wherein said reducing agent is BH₃THF.